The objectives of this project are to study the role of protein kinase and of protein phosphorlation in the regulation of human erythrocyte membrane functions. A cyclic AMP-independent protein kinase, which catalyzes the phosphorylation of casein, phosvitin, spectrin and band 3 (membrane anion transporting protein), has been purified to homgeneity in our laboratory. Attempt will be made to investigate in greater detail the phosphorylation of purified spectrin and band 3 by the homogeneous kinase preparation. Since both spectrin and band 3 are phosphoproteins, their phosphoryl acceptor capacity may be limited; hence it may be necessary to dephosphorlate these proteins if they are to be used as substrates for further studies. The proteolytic fragments containing the phosphorylation sites of these substrates will be partially sequenced. In addition to the phosphorylation of a number of exogenous and endogenous substrates, the kinase can also catalyze self-phosphorylation. The possibility that the phosphoenzyme may be able to transfer its phosphate to casein or spectrin will be investigated. Finally, antibody of the kinase will be prepared and used for further investigating the role of the kinase and protein phosphorylation in membrane processes. The antibody will also be used to screen for membrane abnormalities due to a deficiency or an alteration of the protein kinase.